The long-range goal of the proposed research is better understanding of structures and mechanisms of metal-containing enzymes. We have chosen a number of Ni, Fe, V/Mo, and Mn systems where soft x-ray spectroscopy and x-ray magnetic circular dichroism (XMCD) have special strengths for answering the important issues. As needed, we will also use EXAFS and FT-IR spectroscopy. Our emphasis will be issues of electronic structure and catalytic mechanism that are difficult to address by crystallography. Towards this end, we will also explore cryogenic and photochemical approaches to sample preparation. The general hypotheses to be tested include: [1.] Is the conversion between high-spin and low-spin Ni(II) species important for the reactivity of Ni enzymes. To what extent is the redox activity Ni-based, and when is it more appropriate to describe the electronic changes as centered on the ligands? How does the distribution of spin density compare with the distribution of electron density? What are the structures and activation mechanisms for catalytically inactive precursor forms of Ni enzymes? When is Ni involved in substrate binding, and when is it a 'spectator'? What are the roles of Cu and Zn in these enzymes? [2.] During nitrogen fixation, are V/Mo present as 'spectators' to modulate the catalytic activity of the Fe, or are the key substrate reactions on V/Mo? Are V/Mo magnetically coupled to the rest of the M-center? [3.] How does the electron density change on Mn throughout the catalytic cycle of photosystem ll? How do inhibitors such as F- bind, and how does this change in different S-states? Although the focus of our experiments is primarily at the electronic and molecular level, it is also important to understand the cellular distribution of these trace metals. Recent advances in soft x-ray microscopy allow x-ray tomographic analysis at the 500 A scale. We propose to exploit our knowledge of metalloenzyme soft x-ray spectroscopy to record images that are selective for Fe or Ni. In order to conduct better x-ray experiments, we will make improvements to existing equipment and also develop some new instrumentation. We will build a small cryostat with differential pumping, so that soft x-ray experiments no longer require UHV. We will also fabricate a 36-element contiguous STJ detector for fluorescence detected x-ray absorption measurements.